Doctor of Philosophy, The Ohio State University, 2024, Plant Pathology

Often overshadowed by staple crops, vegetables contain many essential vitamins and minerals and play a key role in global food and nutritional security. However, vegetable production is threatened by a variety of diseases, including bacterial wilt and bacterial spot. My dissertation research utilizes genomic surveys to elucidate the diversity of the bacteria causing these diseases, both globally and locally. Bacterial wilt disease is endemic within the country of Cambodia, causing significant yield losses for Cambodian growers. However, the diversity of the Ralstonia solanacearum Species Complex (RSSC), the causal agent responsible for bacterial wilt disease, has not been defined in Cambodia. Therefore, we conducted a bacterial wilt survey within Cambodia, collecting RSSC isolates from four distinct host plants (tomato, hot pepper, long bean, and bitter gourd) over three locations, for a total of 24 RSSC isolates. We found that all 24 of the Cambodian RSSC isolates belong to phylotype I and are classified as Ralstonia pseudosolanacearum. Through disease progress assays on susceptible hosts, we observed that variation in the Cambodian isolate's ability to cause consistent wilt was dependent on the method of inoculation. Additionally, the Cambodian R. pseudosolanacearum isolates exhibited a wide range of phylogenomic diversity. When comparing the core and accessory genome and the Type III effector profile of the Cambodian isolates, we found that the R. pseudosolanacearum accessory genome better reflected the host of isolation and host range of the isolates compared to the core genome. Altogether, this research provides a glimpse into the RSSC diversity present within Cambodia and insight into R. pseudosolanacearum host range. Bacterial spot disease affects tomato and pepper production worldwide and is caused by a species complex of Xanthomonas bacteria: X. hortorum pv. gardneri, X. euvesicatoria pv. euvesicatoria, X. euvesicatoria pv. perforans, and X. vesicatoria. We (open full item for complete abstract)

Committee: Jonathan Jacobs (Advisor); Mary Rodriguez (Committee Member); Francesca Hand (Committee Member); Sally Miller (Advisor) Subjects: Plant Pathology

Doctor of Philosophy, The Ohio State University, 2019, Horticulture and Crop Science

The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technology has enabled targeted genome modifications in plants through genome editing and gene transcriptional modifications. For high value crops such as soybean [Glycine max (L.) Merr.], CRISPR has the potential to induce targeted genome modifications for crop improvements faster and with higher precision than conventional breeding and transgenic strategies. In its basic form, CRISPR functions through the engineered use of compatible guide RNA and Cas nuclease, which are both simpler and more accessible than presently available alternative targeted genome modification tools such as meganuclease, zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). Nevertheless, all of these genome modification tools need to be customized and calibrated for optimal function in plants. A critical customization for CRISPR in soybean is the use of appropriate promoters for introducing the DNA precursors of guide RNA and Cas nuclease. In addition, CRISPR can also be directed to target soybean promoters, leading to precise promoter modifications and modulation of native gene expression. CHAPTER 1 of this dissertation reviews the important functions and applications of promoters in CRISPR-based plant genome modifications. Next, CHAPTER 2 describes the isolation and characterization of 40 unique promoters from soybean, which broaden the toolbox of genetic regulatory components for genome modifications. CHAPTER 3 describes the adaptation of CRISPR interference (CRISPRi) for interrogating cis-elements within a soybean promoter, resulting in pertinent information for future applications of this transcriptional modification strategy. Lastly, CHAPTER 4 describes multiple applications of soybean promoters to both confirm and improve a CRISPR-based targeted DNA integration strategy in plants, termed homology-independent targeted integration (HITI). Customized, particle bombardment-b (open full item for complete abstract)

Committee: John Finer (Advisor); Jonathan Fresnedo Ramirez (Committee Member); Feng Qu (Committee Member); Eric Stockinger (Committee Member) Subjects: Agriculture; Biology; Botany; Cellular Biology; Molecular Biology; Plant Biology; Plant Sciences; Technology

Doctor of Philosophy, The Ohio State University, 2021, Horticulture and Crop Science

The production of greenhouse ornamental crops relies on extensive inputs of water and chemical fertilizers to produce high-quality plants for consumers. These inputs are both economically and resource expensive, leading to increased concerns of sustainability. In addition, ornamental crops can encounter water and nutrient stress throughout their life span, impacting their health, quality, and resiliency for consumers. Water stress decreases the health and quality of horticulture crops by inhibiting photosynthesis, transpiration, and nutrient uptake, contributing to a reduction in plant size and flower number. The lack of bioavailable nutrients for plant uptake negatively impacts plant metabolism, influencing different aspects of plant growth and development. The effect of both abiotic stresses decreases the salability of crops at retail and can impact consumer success in the landscape. Therefore, it is important that the horticulture industry has sustainable tools to decrease resource-intensive inputs while also increasing plant abiotic stress tolerance, without sacrificing crop quality. Plant growth promoting bacteria (PGPB) can increase plant growth under water and nutrient-limiting conditions by enhancing stress tolerance and increasing nutrient availability, uptake, and assimilation by plants. PGPB colonize their plant host and can stimulate plant growth and stress tolerance through a myriad of different mechanisms. The identification of PGPB for greenhouse ornamental crops will contribute to the formulation of commercial productions that can be implemented into greenhouse production systems for the sustainable production of high-quality and resilient crops. This work outlines the identification, evaluation, and characterization of PGPB for greenhouse ornamental crops subjected to water stress and low-nutrient conditions. A core collection of 45 bacterial isolates was utilized to develop a high-throughput approach for the selection and evaluation of PG (open full item for complete abstract)

Committee: Michelle Jones (Advisor); Christopher Taylor (Committee Member); Ye Xia (Committee Member); Jyan-Chyun Jang (Committee Member) Subjects: Horticulture; Plant Biology; Plant Pathology

Doctor of Philosophy (PhD), Ohio University, 2020, Plant Biology (Arts and Sciences)

Flowering plants produce two different kinds of flowers: chasmogamous, open flowers that promote cross-pollination, and cleistogamous, small mechanically sealed flowers that force self-pollination. Typically, plants produce either chasmogamous or cleistogamous flowers. However, some species contain a chasmogamous/cleistogamous mixed breeding system and develop both flowers at different locations along the plant or at different times of the season. The temporal separation of chasmogamous and cleistogamous flowers is hypothesized to be influenced by the environment. However, the specific environmental cues and genetic mechanisms that signal for the induction and differentiation of each flower type remain largely unknown. To analyze the environmental variables driving separate, seasonal development of chasmogamous and cleistogamous flowers in Viola pubescens, bud counts and measurements of light quantity, canopy cover, photoperiod, temperature, soil moisture, and soil pH were collected over a native population in 2016 and 2017. Statistical analyses confirm that the two flowers develop in response to differing environmental factors and distinguish which factors are significantly associated with chasmogamous versus cleistogamous bud presence, the number of buds produced, and specific threshold values at which each bud type is most likely to develop. To enable genetic investigations, the V. pubescens draft genome was assembled using DNA and RNA sequencing data from eight diverse tissues, which together generated 38,081 gene models including 81 novel cyclotide genes. Gene expression was visualized for each tissue and highlighted transcriptional networks specific to chasmogamous and cleistogamous flowers. Functional annotation of the flower-specific genes indicate that chasmogamous flowers are enriched with genes involved in environmental sensing and repressing precocious development, while cleistogamous flowers contain up-regulated genes involved in DNA topology and microR (open full item for complete abstract)

Committee: Harvey Ballard Jr. (Advisor); Sarah Wyatt (Advisor); David Rosenthal (Committee Member); Ronan Carroll (Committee Chair) Subjects: Bioinformatics; Biology; Developmental Biology; Ecology; Environmental Science; Genetics; Plant Biology; Plant Sciences

Doctor of Philosophy, The Ohio State University, 2020, Translational Plant Sciences

Phytophthora sojae is a destructive oomycete pathogen of soybean [Glycine max (L) Merr], which causes yield losses in many soybean-growing regions and results in worldwide losses in excess of $1 billion. Genetic resistance is the preferred method of managing P. sojae. Resistance is inherited both qualitatively and quantitatively, with both providing crucial elements of genetic resistance. Quantitative disease resistance (QDR) is a complex trait, controlled by many loci and at least 22 genetic mapping studies have been completed, identifying a highly polygenic trait. In this research, we contribute to the understanding of this pathosystem by (1) summarizing the current literature of the P. sojae-soybean pathosystem, (2) mapping QDR loci in diverse soy germplasm to provide novel alleles for breeding programs, (3) testing genomic prediction (GP) to determine which methodology results in the most accurate GP model, (4) and validating the GP methods across diverse germplasm. The results of this dissertation include utilizing genome-wide association analyses to identify 44 QDR loci towards P. sojae, including 14 novel loci. The analyses completed here were among the first to test how accurate GP would be for P. sojae QDR traits. The GP accuracy averaged 0.51 across nine measurements of seedling phenotypes and demonstrated that the accuracy of the GP was relatively independent of methodology; rather the measurement of QDR was the largest factor contributing to differences in accuracy. When GP was completed across a collection of genetically diverse germplasm the accuracy decreased to between 0.14 and 0.43, and though reduced, the accuracy remained high enough to merit further investigation for genomic selection in applied breeding programs. Overall these results have built upon strong research and added to the understanding of the genetic architecture of QDR towards P. sojae, identified novel QDR alleles for breeding programs, and provide an initial estimate how effective (open full item for complete abstract)

Committee: Leah McHale (Advisor); Anne Dorrance (Advisor); Guo-Liang Wang (Committee Member); Christopher Taylor (Committee Member); Eric Stockinger (Committee Member); Aaron Lorenz (Committee Member) Subjects: Genetics; Plant Biology; Plant Pathology; Plant Sciences

Doctor of Philosophy, The Ohio State University, 2019, Plant Pathology

Fungal interactions with plants pose both significant risks and benefits to global economies and ecosystems. As pathogens, fungi consume crops at our expense, and as mutualists and decayers, they maintain the health of fields, forests and soils. A key trait underlying these varied lifestyles is the ability to degrade toxic chemicals produced by plants to defend themselves from fungal attack. However, little is known about the genetic bases of these degradative (i.e., catabolic) mechanisms, or the evolutionary processes that give rise to adaptive catabolism, which has resulted in a fundamental gap in our understanding of how fungi adapt to their plant hosts. One promising approach to address these gaps in our knowledge is the study of metabolic gene clusters (MGCs), which are groups of neighboring genes that encode enzymatic, transporter and regulatory proteins participating in the same or related metabolic pathway. The self-contained nature of MGCs facilitates the discovery of genes encoding adaptive pathways, as well as investigations into the mechanisms shaping their evolution. Yet the extent to which catabolic genes form MGCs is unknown, largely due to a lack of tools suitable for their identification. The primary research objectives of this dissertation are thus twofold: to first develop computational tools for the identification of MGCs encoding the degradation of plant defense chemicals, and to then characterize the MGCs identified by these tools using phylogenetic and functional genetic analyses in order to elucidate the evolutionary processes driving fungal catabolic adaptation to plant tissues. In Chapter 1, I synthesize what is currently known about catabolic MGCs and their contributions to fungal ecological adaptation, with a focus on the evolutionary forces driving their assembly, maintenance and dispersal in fungal populations. In Chapter 2, I review the impact of one of these forces, horizontal gene transfer, on the evolution of eukaryotic microbial (open full item for complete abstract)

Committee: Jason Slot (Advisor); Ana Alonso (Committee Member); Pierluigi Bonello (Committee Member); Laura Kubatko (Committee Member) Subjects: Plant Pathology

Doctor of Philosophy, The Ohio State University, 2018, Plant Pathology

Salmonellosis cases caused by Salmonella enterica through pre-harvest contamination of fresh produce represent a risk to human health worldwide; however, little is known about the interactions between Salmonella, phytopathogens, environment, and the plant host contributing to this food safety issue. Furthermore, the control of Salmonella from “farm to fork” is challenging due to the development of resistance mechanisms towards current control methods and restrictions on use of antimicrobials imposed by regulatory agencies. We investigated the effects of specific environmental conditions on the persistence and dissemination of Salmonella enterica subsp. enterica serotype Typhimurium (S. Typhimurium) following artificial contamination of `Tiny Tim' tomato plants. We found that higher temperatures (30°C day/25°C night) reduced the persistence of S. Typhimurium in the phyllosphere compared to lower temperatures (20°C day/15°C night) when plants were sprayed on the leaves with a S. Typhimurium -contaminated solution. Wounding cotyledons with contaminated tools increased S. Typhimurium persistence and internalization in planta compared to spray inoculation. Low relative humidity enhanced the dissemination of Salmonella into non-inoculated plant tissues. S. Typhimurium was detected in the root systems for at least 98 days-post inoculation. Further, we showed that splice-grafting (`Celebrity' with 'MaxiFort') is a major risk for the internalization and long-term survival of S. Typhimurium inside the tomato plant. S. Typhimurium was detected in the root system for over 137 days if at least 5 x 10^3 colony-forming units were introduced during grafting. The survival of S. Typhimurium in tomato foliage was also affected by the presence of phytopathogens, the genotype of S. Typhimurium and tomato variety used. We found that rfbV, involved in O antigen synthesis, might be essential for S. Typhimurium persistence in inoculated tomato plants and especially in `Tiny Tim' plants (open full item for complete abstract)

Committee: Gireesh Rajashekara (Advisor); Sally Miller (Advisor); Laurence Madden (Committee Member); Christopher Taylor (Committee Member); Corey Nislow (Committee Member) Subjects: Agriculture; Bioinformatics; Biology; Environmental Health; Molecular Biology; Plant Pathology; Public Health

Doctor of Philosophy, The Ohio State University, 2018, Evolution, Ecology and Organismal Biology

Most broadly, this study aimed to develop a better understanding of how organisms evolve novel functions and traits, and examine how seemingly complex adaptive trait syndromes can convergently evolve. As an ideal example of this, the carnivorous plants were chosen. This polyphyletic grouping contains taxa derived from multiple independent evolutionary origins, in at least five plant orders, and has resulted in striking convergence of niche and morphology. First, a database study was performed, with the goal of understanding the evolutionary trends that impact carnivorous plants as a whole. Using carnivorous and non-carnivorous plant genomes available from GenBank. An a priori list of Gene Ontology-coded functions implicated in plant carnivory by earlier studies was constructed via literature review. Experimental and control samples were tested for statistical overrepresentation of these functions. It was found that, while some functions were significant in some taxa, there was no overall shared signal of plant carnivory, with each taxon presumably having selected for a different subset of these functions. Next, analyses were performed that targeted Sarracenia alata specifically. A reference genome for S. alata was assembled using PacBio, Illumina, and BioNano data and annotated using MAKER-P with additional preliminary database filtration. From these, it was found that Sarracenia alata possesses significant and substantial overrepresentation of genes with functions associated with plant carnivory, at odds with the hypothesis that the plant primarily relies on symbioses. Finally, pitcher fluid was collected from S. alata in the field. RNA was extracted from the fluid, sequenced via Illumina, and assembled with Trinity. Sequences were sorted into host plant and microbiome based on BLAST match to the S. alata reference genome. It was found that, while S. alata contributes two-thirds of the transcripts, these encode no digestive enzymes and a very limited set o (open full item for complete abstract)

Committee: Bryan Carstens Ph.D. (Advisor); Marymegan Daly Ph.D. (Committee Member); Zakee Sabree Ph.D. (Committee Member); Andrea Wolfe Ph.D. (Committee Member) Subjects: Bioinformatics; Biology; Botany

Doctor of Philosophy, The Ohio State University, 2015, Entomology

Host-plant resistant (HPR) crop varieties provide effective and inexpensive pest control through the expression of natural resistance genes. However, the durability of HPR is often short lived as insect populations evolve virulence, i.e. the ability to bypass these natural defenses. The evolutionary and genetic mechanisms that produce virulence govern its proliferation are largely unstudied. Understanding these mechanisms could provide valuable insight into how virulence emerges and spreads within populations, allowing for the development of more effective virulence management tactics. In this dissertation I have integrated population genetic and genomic analyses with field ecology to investigate the evolutionary and genetic roots of virulence in the soybean aphid (Aphis glycines), and how this information can be applied to increase the durability of HPR soybean. Population genetic analysis in chapter two revealed no significant genetic differentiation between avirulent and virulent biotypes. This pattern is consistent with the uninhibited gene flow and long distance migration found in past studies, and suggests biotypes regularly interbreed. These patterns do not align with a simple gene-for-gene interaction, but are indicative of polygenic or non-genetic adaptation. This prediction was clarified in chapter three where population genomic analyses revealed regions of the genome under divergent selection between avirulent and virulent biotypes clustered both spatially and functionally, in agreement with the polygenic model. Further, transposable elements and transcription/translation regulators were over represented within these divergent genomic regions, suggesting their probable role in virulence evolution. Chapter four describes that construction of a draft genome assembly of A. glycines. The final assembly represents the smallest known aphid genome (317.2Mb) and is highly continuous and complete with a length of 302.9Mb (95.5% of predicted genome) spread (open full item for complete abstract)

Committee: Andrew Michel Ph.D. (Advisor); Mary Gardiner Ph.D. (Committee Member); McHale Leah Ph.D. (Committee Member); Hoy Casey Ph.D. (Committee Member) Subjects: Entomology

Master of Science, The Ohio State University, 2015, Horticulture and Crop Science

Phytophthora root and stem rot of soybean (Glycine max) is caused by the oomycete pathogen Phytophthora sojae. This disease can be controlled by genetic resistance, but can cause devastating yield losses in fields planted with susceptible soybean cultivars and results in losses of around $300 million annually in the US. Partial resistance is considered to be more durable against P. sojae than race-specific resistance conferred by Rps genes and is theoretically effective against all races of this pathogen. Evaluation of a historical set of public cultivars representing 80 years of soybean breeding indicated that there have been genetic gains for partial resistance; however, these gains may have begun to plateau in the 1970s to early 1980s. Cultivars developed in Ohio generally have high levels of partial resistance to P. sojae; however, there is little known about the genetic regions associated with the partial resistance. Further improvement of increasing partial resistance could be achieved through the introgression of known quantitative trait loci (QTL) from plant introductions from the Republic of Korea (South Korea), which contain high levels of partial resistance. From an analysis of 1,398 plant introductions with a wide range of phenotypic expression of resistance, sixteen single nucleotide polymorphisms (SNPs) were associated with partial resistance to P. sojae. These SNPs were located in three genomic regions, or QTL, on chromosomes 3, 13, and 19. The QTL on chromosome 19 represented a novel locus, whereas the QTL on chromosomes 3 and 13 were coincident with previously identified QTL for partial resistance and/or Rps genes. In contrast, a genome-wide association study carried out in Ohio breeding lines was unable to detect any significant marker-trait associations, limiting the ability to use marker assisted selection to improve partial resistance in this population. However, genomic selection (GS) was shown to be a promising means of selection, with (open full item for complete abstract)

Committee: Leah McHale Dr. (Advisor); Anne Dorrance Dr. (Advisor); Clay Sneller Dr. (Committee Member) Subjects: Agronomy; Horticulture; Plant Pathology; Plant Sciences

Doctor of Philosophy, The Ohio State University, 2014, Horticulture and Crop Science

The genus Phlox is a staple of gardens worldwide that includes species admired for beauty and versatility in gardens, constructed landscapes, containers, and as cut flowers. Extensive breeding and selection has occurred in three primary species: Phlox drummondii, P. paniculata, and P. subulata, but the genus includes other species with ornamental value. Phlox L. (Polemoniaceae) includes approximately 65 species primarily endemic to North America; 20-23 species occur in the eastern U.S. and 40-45 in the west. The eastern species are a polymorphic group organized into 6 subsections that include the three main cultivated species and up to 20 related, rarely cultivated species. Thus, the species diversity of Phlox has barely been applied for ornamental use. The widespread availability of diverse germplasm can contribute not only to new cultivated forms but also to a greater understanding of species diversity and relationships. Such interest has made Phlox a priority genus for conservation at the Ornamental Plant Germplasm Center. This work describes the development, partial characterization, and manipulation of Phlox germplasm. Phlox germplasm collection development began in 2010 with an effort to collect all eastern species from natural populations throughout their native ranges; 187 accessions were collected from wild populations of 22 eastern species during a series of expeditions. Another 166 accessions were of cultivated origin; these were used for comparison to wild-collected material. The 353 accessions represent the most comprehensive germplasm collection of Phlox to date. This germplasm was first characterized by estimation of genome size using flow cytometry and ploidy estimates by chromosome counts. Genome size was surveyed in 287 accessions; 165 accessions were of wild origin and the rest were cultivars. Most accessions were diploid, but genome size was variable; both tetraploid and hexaploid populations were found in a species where it had n (open full item for complete abstract)

Committee: Pablo Jourdan (Advisor); Mark Bennett (Committee Member); David Francis (Committee Member); John Freudenstein (Committee Member) Subjects: Horticulture